Angiogenesis, or the process of producing new blood vessels in the body, is a key step in a number of biological responses to injury, stroke, and tumor formation. Upon angiogenic stimulation, endothelial cells re-enter the cell cycle, migrate, withdraw from the cell cycle and subsequently differentiate again to form new vessels that are functionally adapted to their tissue environment. Endothelial cells undergoing angiogenesis degrade the underlying basement membrane and migrate, forming capillary sprouts that project into the perivascular stroma. Ausprunk, et al., Microvasc. Rev., 14:51–65 (1977). In most cells angiogenesis is under tight control by a series of oxygen-sensitive proteins that act in concert to prevent undue blood vessel formation.
A key protein mediator of angiogenesis is vascular endothelial growth factor (VEGF), a potent stimulator of blood vessel growth. Vascular endothelial growth factor is a dimeric glycoprotein with two 23 kDa subunits that are linked by disulfide bonds. Vascular endothelial growth factor is believed to influence the mobilization of intracellular calcium, the induction of plasminogen activator, the synthesis of plasminogen activator inhibitor-1, the stimulation of hexose transport in endothelial cells, and the promotion of monocyte migration in vitro. Four VEGF isoforms, encoded by distinct mRNA splice variants, appear to be equally capable of stimulating mitogenesis in endothelial cells. Each isoform has a different affinity for cell surface proteoglycans, which behave as low affinity receptors for VEGF. The 121 and 165 amino acid isoforms of VEGF (VEGF121 and VEGF165) are secreted in a soluble form, whereas the isoforms of 189 and 206 amino acid residues remain cell surface associated and have a strong affinity for heparin.
Two high affinity receptors for vascular endothelial growth factor have been characterized. These are VEGFR-1/Flt-1 (fms-like tyrosine kinase-1) and VEGFR-2/Kdr/Flk-1 (kinase insert domain containing receptor/fetal liver kinase-1). Such receptors are classified as being in the PDGF-receptor family, but they have seven rather than five immunoglobulin-like loops in their extracellular domain and they possess a longer kinase insert than normally observed in this family. The expression of VEGF receptors occurs mainly in vascular endothelial cells, although some may be present on monocytes and melanoma cells. Only endothelial cells have been reported to proliferate in response to VEGF, and endothelial cells from different sources may show different responses.
The pattern of VEGF expression suggests its involvement in the development and maintenance of the normal vascular system and in tumor angiogenesis. During murine development, the entire 7.5 day post-coital (p.c.) endoderm expresses VEGF and the ventricular neuroectoderm produces VEGF at the capillary ingrowth stage. See Breier, et al., Development, 114:521–523 (1992). On day two of quail development, the vascularized area of the yolk sac as well as the whole embryo show expression of VEGF. In addition, epithelial cells next to fenestrated endothelia in adult mice show persistent VEGF expression, suggesting a role in the maintenance of this specific endothelial phenotype and function.
Tumor cells often express VEGF at levels three to ten times higher than normal cells. Tumor growth and metastasis have also been shown to be angiogenesis dependent. Folkman, et al., J. Biol. Chem., 267:10931–10934 (1992). Consequently, a need exists for agents that can inhibit the expression and/or activity of VEGF.